U.S. patent application number 10/692105 was filed with the patent office on 2004-09-02 for process for the production of a reversibly inactive acidified plasmin composition.
Invention is credited to Bradley, Rita T., Cook, Scott A., Dadd, Christopher A., Kent, Jonathan D., Korneyeva, Marina N., Novokhatny, Valery V., Rebbeor, James F., Stenland, Christopher J., Strauss, Jonathan S., Terry, Jarrett C., Yuziuk, Jeffrey A..
Application Number | 20040171103 10/692105 |
Document ID | / |
Family ID | 46300182 |
Filed Date | 2004-09-02 |
United States Patent
Application |
20040171103 |
Kind Code |
A1 |
Bradley, Rita T. ; et
al. |
September 2, 2004 |
Process for the production of a reversibly inactive acidified
plasmin composition
Abstract
Disclosed is both a process for producing a reversibly inactive
acidified plasmin by activating plasminogen and a process for
producing a purified plasminogen. The produced plasmin is isolated
and stored with a low pH-buffering capacity agent to provide a
substantially stable formulation. The purified plasminogen is
typically purified from a fraction obtained in the separation of
immunoglobulin from Fraction II+III chromatographic process and
eluted at a low pH. The reversibly inactive acidified plasmin may
be used in the administration of a thrombolytic therapy.
Inventors: |
Bradley, Rita T.; (Cary,
NC) ; Cook, Scott A.; (Garner, NC) ; Dadd,
Christopher A.; (Holly Springs, NC) ; Kent, Jonathan
D.; (Holly Springs, NC) ; Korneyeva, Marina N.;
(Raleigh, NC) ; Novokhatny, Valery V.; (Raleigh,
NC) ; Rebbeor, James F.; (Garner, NC) ;
Stenland, Christopher J.; (Cary, NC) ; Strauss,
Jonathan S.; (Walnut Creek, CA) ; Terry, Jarrett
C.; (Raleigh, NC) ; Yuziuk, Jeffrey A.;
(Garner, NC) |
Correspondence
Address: |
WOMBLE CARLYLE SANDRIDGE & RICE, PLLC
P.O. BOX 7037
ATLANTA
GA
30357-0037
US
|
Family ID: |
46300182 |
Appl. No.: |
10/692105 |
Filed: |
October 23, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10692105 |
Oct 23, 2003 |
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10143156 |
May 10, 2002 |
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10143156 |
May 10, 2002 |
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PCT/US00/42143 |
Nov 13, 2000 |
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PCT/US00/42143 |
Nov 13, 2000 |
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09438331 |
Nov 13, 1999 |
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6355243 |
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Current U.S.
Class: |
435/68.1 |
Current CPC
Class: |
A61K 47/12 20130101;
A61K 47/183 20130101; A61K 47/20 20130101; A61K 47/26 20130101;
C12N 9/6435 20130101; A61K 38/484 20130101; A61K 38/4833 20130101;
A61K 47/02 20130101; C12Y 304/21007 20130101 |
Class at
Publication: |
435/068.1 |
International
Class: |
C12P 021/06 |
Claims
What is claimed is:
1. A method for purifying plasmin comprising: cleaving a
plasminogen in the presence of a plasminogen activator to yield an
active plasmin; substantially removing the plasminogen activator
from the active plasmin by binding the active plasmin to an active
plasmin-specific absorbent material to form a bound plasmin, and
eluting the bound plasmin with an excipient solution having a pH
from about 2.5 to about 9.0 to form a plasmin solution; and
buffering the plasmin solution with a low pH, low buffering
capacity agent to form a reversibly inactive acidified plasmin.
2. The method of claim 1, wherein the excipient solution has a pH
from about 4.0 to about 7.5.
3. The method of claim 1, wherein the excipient solution has a pH
of about 6.0.
4. The method of claim 1, wherein the active plasmin-specific
absorbent material comprises benzamidine.
5. The method of claim 1, wherein the plasminogen activator is
further removed by hydrophobic interaction.
6. The method of claim 1, further comprising nanofiltration of the
plasmin solution.
7. The method of claim 6, wherein the nanofiltration is carried out
using a filter membrane characterized by an average pore size of
approximately 15 nm.
8. The method of claim 1, wherein the plasminogen is cleaved in the
presence of at least one excipient that is an omega-amino acid.
9. The method of claim 1, wherein the plasminogen is cleaved in the
presence of at least one omega-amino acid selected from the group
consisting of lysine, epsilon amino caproic acid, tranexamic acid,
poly lysine, arginine, and combinations thereof.
10. The method of claim 1, wherein the plasmin is eluted in a
solution comprising at least one salt, the solution having a
conductivity from about 5 mS to about 100 mS.
11. The method of claim 10, wherein the at least one salt is sodium
chloride.
12. The method of claim 11, wherein the sodium chloride is present
at a concentration of from about 50 mM to about 1000 mM.
13. The method of claim 11, wherein the sodium chloride is present
at a concentration of about 150 mM.
14. The method of claim 1, wherein the plasminogen is cleaved using
a catalytic concentration of a plasminogen activator that is
selected from the group consisting of immobilized plasminogen
activators, soluble plasminogen activators, and combinations
thereof.
15. The method of claim 1, wherein the plasminogen activator is
selected from the group consisting of streptokinase, urokinase, tPA
and combinations thereof.
16. The method of claim 1, wherein the plasminogen activator is
soluble streptokinase.
17. The method of claim 1, wherein the plasminogen activator is
immobilized on a solid support medium comprising SEPHAROSE.
18. The method of claim 1, wherein the low pH, low buffering
capacity agent comprises a component selected from the group
consisting of an amino acid, a derivative of at least one amino
acid, an oligopeptide which includes at least one amino acid, and
combinations thereof.
19. The method of claim 1, wherein the low pH, low buffering
capacity agent comprises a component selected from the group
consisting of acetic acid, citric acid, hydrochloric acid,
carboxylic acid, lactic acid, malic acid, tartaric acid, benzoic
acid, serine, threonine, methionine, glutamine, alanine, glycine,
isoleucine, valine, alanine, aspartic acid, derivatives thereof,
and combinations thereof.
20. The method of claim 1, wherein the buffer is present in the
reversibly inactive acidified plasmin at a concentration at which
the pH of the acidified plasmin is raised to neutral pH by adding
serum in an amount no more than about 5 times the volume of the
acidified plasmin.
21. The method of claim 1, wherein the reversibly inactive
acidified plasmin solution has a pH between about 2.5 to about
4.
22. The method of claim 1, further including stabilizing the
reversibly inactive acidified plasmin by adding a stabilizing agent
selected from the group consisting of a polyhydric alcohol,
pharmaceutically acceptable carbohydrates, salts, glucosamine,
thiamine, niacinamide, and combinations thereof.
23. The method of claim 22, wherein the salts are selected from the
group consisting of sodium chloride, potassium chloride, magnesium
chloride, calcium chloride and combinations thereof.
24. The method of claim 1, further including stabilizing the
reversibly inactive acidified plasmin by adding a sugar or sugar
alcohol selected from the group consisting of glucose, maltose,
mannitol, sorbitol, sucrose, lactose, trehalose, and combinations
thereof.
25. A method for purifying plasmin comprising: cleaving a
plasminogen using a catalytic concentration of a plasminogen
activator to yield an active plasmin; binding the active plasmin to
an active plasmin-specific absorbent material to form a bound
plasmin; and eluting the bound plasmin with a substantially neutral
pH excipient solution to form a final plasmin solution which is
substantially free of degraded plasmin.
26. The method of claim 25, wherein the activated plasmin solution
is stabilized by the addition of at least one excipient selected
from the group consisting of omega-amino acids and salts.
27. The method of claim 26, wherein the at least one excipient is
an omega-amino acid selected from the group consisting of lysine,
epsilon amino caproic acid, tranexamic acid, poly lysine, arginine,
analogues thereof and combinations thereof.
28. The method of claim 25, further comprising filtering out amino
acids from the final plasmin solution.
29. The method of claim 25, further comprising adding a low pH, low
buffering capacity agent to the final plasmin solution to form a
reversibly inactive acidified plasmin.
30. The method of claim 29, further comprising adjusting the pH of
the reversibly inactive acidified plasmin to a pH between about 2.5
to about 4.
31. The method of claim 25, further comprising adding a stabilizer
to the final plasmin solution.
32. The method of claim 31, wherein the stabilizer is selected from
the group consisting of amino acids, salts or combinations
thereof.
33. A process for the purification of plasminogen from a plasma
source comprising: extracting plasminogen from a plasma paste
fraction with a buffer solution at a pH in a range from about 3.5
to 10.5 and collecting the plasminogen-containing solution; adding
polyethylene glycol, metal oxide, ammonium sulfate, or a
combination thereof to the plasminogen-containing buffer solution
to precipitate impurities; separating the precipitated impurities
from the effluent containing plasminogen; and adding the effluent
containing plasminogen to a plasminogen-specific absorbent
material.
34. The process of claim 33, further comprising subjecting the
plasminogen-containing solution to cation-exhange chromatography or
ultrafiltration/diafiltration prior to addition to the
plasminogen-specific absorbent material.
35. The process of claim 34, wherein the solution is subjected to
cation-exchange chromatography.
36. The process of claim 33, wherein the plasma source is derived
from Fraction II+III of Cohn plasma fractionation process.
37. The process of claim 33, wherein the buffer solution for
plasminogen extraction is at a pH in a range from about 7.0 to
about 10.5.
38. The process of claim 33, wherein about 1% to about 10% w/w
polyethylene glycol or 80 g/L to 120 g/L ammonium sulfate is
added.
39. The process of claim 33, wherein a particulate metal oxide is
added.
40. The process of claim 39, wherein the particular metal oxide is
silicon dioxide.
41. The process of claim 40, wherien the silicon dioxide is fumed
silica.
42. The process of claim 41, wherein the fumed silica is added in
an amount from about 0.1% to about 1.0% by weight of the
plasminogen-containing buffer solution.
43. The process of claim 41, wherein the fumed silica is added in
an amount from about 0.25% to about 0.5% by weight of the
plasminogen-containing buffer solution.
44. The process of claim 33, further comprising adding a
plasminogen solubility enhancer.
45. The process of claim 44, wherein the plasminogen solubility
enhancer is selected from the group of excipients consisting of
lysine, epsilon amino caproic acid, tranexamic acid, poly lysine,
arginine, combinations thereof and analogues thereof.
46. The process of claim 44, further comprising removing the
plasminogen solubility enhancer.
47. The process of claim 33, wherein the eluted plasminogen is
treated at a pH between about 3 and about 4.
48. The process of claim 33, further comprising stabilizing
plasminogen during pH adjustment from about 3 to neutral by adding
excipients prior to pH adjustment.
49. The process of claim 33, further comprising removing or
inactivating pathogens.
50. The process of claim 49, wherein removing pathogens includes
inactivating viral pathogens and removing TSE pathogens.
51. The process of claim 49, wherein viruses are removed or
inactivated by the steps selected from the group consisting of heat
treatment, caprylate addition, solvent detergent addition,
nanofiltration and combinations thereof.
52. The process of claim 51, wherein TSE are removed by the steps
selected from the group consisting of PEG precipitation, addition
of a particulate metal oxide, depth filtration, nanofiltration, and
combinations thereof.
53. The process of claim 33, wherein the plasminogen-specific
absorbent material comprises a lysine affinity resin.
Description
[0001] This is a continuation-in-part of U.S. application Ser. No.
10/143,156, filed May 10, 2002, itself a continuation of
International Application PCT/US00/42143 filed Nov. 13, 2000 and
published in English on May 25, 2001, itself a continuation-in-part
of U.S. application Ser. No. 09/438,331, filed Nov. 13, 1999 (now
U.S. Pat. No. 6,355,243, issued Mar. 12, 2002).
FIELD OF THE INVENTION
[0002] The present invention relates generally to a method of
producing plasmin and more particularly to a method of purifying
and isolating the plasmin under conditions which stabilize against
degradation.
BACKGROUND
[0003] Fibrin is a white insoluble fibrous protein formed from
fibrinogen by the action of thrombin. In the clotting of blood,
fibrin forms the structural scaffold of a thrombus, which is a clot
of blood formed within a blood vessel that remains attached to its
place of origin. Under normal conditions the blood clotting system
is maintained in equilibrium and the fibrin deposits are dissolved
by the fibrinolytic enzyme system. Unfortunately, events such as
vascular damage, activation/stimulation of platelets, and
activation of the coagulation cascade may disturb the equilibrium,
which can result in thrombosis or the blockage of a blood vessel by
a blood clot.
[0004] Intravascular thrombosis is one of the most frequent
pathological events accounting for greater than 50% of all deaths
as well as a variety of other serious clinical problems. Most
spontaneously developing vascular obstructions are due to the
formation of intravascular blood clots, also known as thrombi.
Small fragments of a clot may detach from the body of the clot and
travel through the circulatory system to lodge in distant organs
and initiate further clot formation. Myocardial infarction,
occlusive stroke, deep venous thrombosis (DVT) and peripheral
arterial disease are well-known consequences of thromboembolic
phenomena.
[0005] Plasminogen activators are currently the favored agents
employed in thrombolytic therapy, all of which convert plasminogen
to plasmin and promote fibrinolysis by disrupting the fibrin matrix
(M. A. Creager and V. J. Dzau, Vascular Diseases of the
Extremities, ppgs. 1398-1406 in Harrison's Principles of Internal
Medicine, 14.sup.th ed., Fauci et al, editors, McGraw-Hill Co., New
York, 1998; the contents of which is incorporated herein by
reference in its entirety).
[0006] The most widely used plasminogen activators include a
recombinant form of tissue-type plasminogen activator (tPA),
urokinase (UK) and streptokinase (SK), as well as a new generation
of plasminogen activators selected for improved pharmacokinetics
and fibrin-binding properties. All of these plasminogen activators,
however, by virtue of their mechanism of action, act indirectly and
require an adequate supply of their common substrate, plasminogen,
at the site of the thrombus to effect lysis.
[0007] UK and tPA convert plasminogen to plasmin directly by
cleaving the Arg.sup.560-Val.sup.561 peptide bond. The resulting
two polypeptide chains of plasmin are held together by two
interchain disulfide bridges. The light chain of 25 kDa carries the
catalytic center and is homologous to trypsin and other serine
proteases. The heavy chain (60 kDa) consists of five triple-loop
kringle structures with highly similar amino acid sequences. Some
of these kringles contain so-called lysine-binding sites that are
responsible for plasminogen and plasmin interaction with fibrin,
.alpha.2-antiplasmin or other proteins. SK and staphylokinase
activate plasminogen indirectly by forming a complex with
plasminogen, which subsequently behaves as a plasminogen activator
to activate other plasminogen molecules by cleaving the
arginyl-valine bond.
[0008] Although thrombolytic drugs, such as tissue plasminogen
activator (tPA), streptokinase, and urokinase, have been
successfully employed clinically to reduce the extent of a
thrombotic occlusion of a blood vessel, it appears that serious
limitations persist with regard to their use in current
thrombolytic therapy. For example, because the activation of
plasminogen by tPA is fibrin dependent for full proteolytic
activity to be realized (Haber et al. 1989), excessive bleeding may
result as a side effect of its use. Other adverse sequelae
associated with the use of these thrombolytic agents include
myocardial infarction, occlusive stroke, deep venous thrombosis and
peripheral arterial disease.
[0009] Additionally, the known plasminogen activators currently
used suffer from several limitations that impact their overall
usefulness in the elimination of a thrombus. For example, at best,
the use of current thrombolytic therapy results in restored
vascular blood flow within 90 min in approximately 50% of patients,
while acute coronary re-occlusion occurs in roughly 10% of
patients. Coronary recanalization requires on average 45 minutes or
more, and intracerebral hemorrhage occurs in 0.3% to 0.7% of
patients. Residual mortality is at least 50% of the mortality level
in the absence of thrombolysis treatment.
[0010] A different approach to avoid the problems associated with
the systemic administration of a plasminogen activator to generate
sufficient plasmin at the site of the thrombus, is to directly
administer the plasmin itself to the patient.
[0011] In U.S. Pat. No. 5,288,489, Reich et al., disclose a
fibrinolytic treatment that includes parenterally introducing
plasmin into the body of a patient. The concentration and time of
treatment were selected to be sufficient to allow adequate active
plasmin to attain a concentration at the site of an intravascular
thrombus that is sufficient to lyse the thrombus or to reduce
circulating fibrinogen levels. However, the necessity of generating
the plasmin from plasminogen immediately prior to its introduction
into the body is also disclosed.
[0012] In contrast, U.S. Pat. No. 3,950,513 to Jenson teaches that
plasmin compositions may be stabilized at pH 7.0 by including a
physiological non-toxic amino acid. This method dilutes stock
plasmin solutions stored at low pH with the neutralizing amino acid
immediately prior to administration. There are advantages, however,
in maintaining low pH of the plasmin composition as long as
possible to minimize autodegradation. Ideally, the plasmin will be
retained at a low pH until encountering the target fibrin.
[0013] Yago et al. disclose plasmin compositions useful as a
diagnostic reagent in U.S. Pat. No. 5,879,923. The compositions of
Yago et al. comprise plasmin and an additional component which may
be 1) an oligopeptide comprising at least two amino acids, or 2) at
least two amino acids, or 3) a single amino acid and a polyhydric
alcohol. However, the compositions of Yago et al. are formulated at
a neutral pH to maintain the enzymatic activity of plasmin.
[0014] Plasmin as a potential thrombolytic agent has numerous
technical difficulties. These difficulties include the challenge of
preparing pure plasmin that is free of all functional traces of the
plasminogem activator used to convert plasmin from its inactive
precursor, plasminogen. Preparations of plasmin are typically
extensively contaminated by plasminogen activator, streptokinase or
urokinase and the thrombolytic activity was, therefore, attributed
to the contaminating plasminogen activators rather than to plasmin
itself. The contaminating plasminogen activators could also trigger
systemic bleeding other than at the targeted site of thrombosis. A
drawback of streptokinase containing plasmin preparations is that
streptokinase can cause adverse immune reactions including fever
and anaphylactic shock.
[0015] One of the more important technical factors limiting
clinical use of plasmin is that plasmin, as a serine protease with
broad specificity, is highly prone to autodegradation and loss of
activity. This circumstance provides severe challenges to the
production of high-quality plasmin, to the stable formulation of
this active protease for prolonged periods of storage prior to use,
and to safe and effective administration of plasmin to human
patients suffering from occlusive thrombi. Thus, there is need for
a method of producing stable plasmin.
SUMMARY
[0016] The present invention provides for both a process for
producing a reversibly inactive acidified plasmin by activating
plasminogen and a process for producing a purified plasminogen. The
produced plasmin is isolated and stored in a low pH, low buffering
capacity agent to provide a substantially stable formulation. The
purified plasminogen is typically purified from a fraction obtained
in the separation of immunoglobulin from Cohn Fractions II+III.
(see, e.g., Cohn, E. J., et al., J. Amer. Chem. Soc., 68:459
(1946); E. J. Cohn, U.S. Pat. No. 2,390,074; and Oncley, et al., J.
Amer. Chem. Soc., 71:541 (1949), the entire disclosures of which
are hereby incorporated by reference herein) by affinity
chromatography with an elution at a low pH. The reversibly inactive
acidified plasmin may be used in the administration of a
thrombolytic therapy.
[0017] Briefly, the method for purifying plasmin comprises cleaving
a plasminogen in the presence of a plasminogen activator to yield
an active plasmin and removing the plasminogen activator from the
active plasmin to form a plasmin solution. A low pH, low buffering
capacity agent can then be added to the final plasmin solution to
form a reversibly inactive acidified plasmin. The final plasmin
solution may be buffered to a pH of between about 2.5 to about
4.
[0018] The plasminogen activator can be removed from the active
plasmin by binding the active plasmin to an active plasmin-specific
absorbent material to form a bound plasmin. One such active
plasmin-specific absorbent material can comprise benzamidine. Once
bound, the active plasmin can be eluted with a low pH solution to
form a final plasmin solution. Plasminogen activator may also be
further removed by hydrophobic interaction.
[0019] A further method of purifying plasmin comprises cleaving
plasminogen to yield an active plasmin and binding the active
plasmin to an active plasmin-specific absorbent material to form a
bound plasmin. The bound plasmin can be eluted with a substantially
neutral pH solution to form a final plasmin solution which is
substantially free of degraded plasmin. The substantially neutral
pH solution can comprise excipients such as omega-amino acids and
salts that are typically filtered out or otherwise removed from the
final plasmin. The final plasmin may also be buffered with a low
pH, low buffering capacity agent.
[0020] The process for the purification of plasminogen from a
plasma source includes the steps of adding the plasminogen
containing solution to a plasminogen-specific absorbent material
and then eluting the plasminogen from the plasminogen-specific
absorbent material at a pH of between about 1 to about 4. The
purified plasminogen is then collected as an eluate. Additionally,
the process may include methods for the purification of micro- or
mini-plasmin(ogen) or other truncated or modified forms of
plasmin(ogen).
[0021] Thus, a process is now provided that successfully addresses
the shortcomings of existing processes and provides distinct
advantages over such processes. Additional objects, features, and
advantages of the invention will become more apparent upon review
of the detailed description set fourth below when taken in
conjunction with the accompanying drawing figures, which are
briefly described as follows.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 graphically depicts the effect of on plasminogen
recovery and lipid removal from CCI filtrate I through polyethylene
glycol (PEG) precipitation/depth filtration;
[0023] FIG. 2 graphically depicts nephelometry data for CCI extract
and the subsequent filtrates I and II;
[0024] FIG. 3 depicts a gel of Coomassie stained reduced SDS-PAGE
(10-20% Tris-Glycine) of CCI extract, filtrates and UF/DF
retentate;
[0025] FIG. 4 depicts a Coomassie stained reduced SDS-PAGE (10-20%
Tris-Glycine) of lysine SEPHAROSE 4B affinity purification of
Plasminogen (Pmg);
[0026] FIG. 5 graphically depicts a lysine SEPHAROSE 4B
chromatogram for the affinity purification of Pmg;
[0027] FIG. 6 depicts a Coomassie stained reduced SDS-PAGE (10-20%
Tris-Glycine) of pH adjustment of the lysine SEPHAROSE 4B eluate
(Pmg) with and without epsilon amino caproic acid (.epsilon.-ACA or
EACA) present;
[0028] FIG. 7 graphically represents streptokinase activation
solution stability following 0.5 M NaCl, 0.25 M .epsilon.-ACA
stop;
[0029] FIG. 8 graphically represents benzamidine SEPHAROSE 6B
chromatogram for the affinity purification of SK activated Plasmin
(Pm);
[0030] FIG. 9 depicts a Coomassie stained reduced SDS-PAGE (10-20%
Tris-Glycine) of benzamidine SEPHAROSE 6B purified Pm;
[0031] FIG. 10 graphically depicts the hydrophobic interaction
chromatography (Octyl-SEPHAROSE 4 FF) chromatogram for the removal
of streptokinase; and
[0032] FIG. 11 depicts a non-reduced SDS PAGE and anti-SK Western
Blot.
[0033] FIG. 12 depicts Western blots illustrating the clearance of
prion proteins by including fumed silica during purification of
plasminogen.
DETAILED DESCRIPTION
[0034] The present invention comprises both a method for producing
a reversibly inactive acidified plasmin in combination with low pH,
low buffering capacity agent and a method for the purification of
plasminogen from a plasma source. The inactive acidified plasmin
solution may also include a stabilizer in addition to being
inactivated in buffered solution. The process for purifying
plasminogen provides for both inactivation and removal of pathogens
and the elution of the plasminogen at a low pH. The inactive
acidified plasmin preparation can be used in the administration of
a thrombolytic therapy.
[0035] Purification of Plasminogen
[0036] The present invention includes both a process for the
purification of plasminogen and plasmin and concurrently, methods
for the inactivation and removal of viral and Transmissible
Spongiform Encephalopathies (TSE) contaminants during these
processes. The terms "TSE" or "TSE contaminants" and "pathogenic
prion protein" are used interchangeably herein unless specifically
noted. The starting material, plasminogen, can be purified from
Cohn Fraction II+III paste by affinity chromatography on
Lys-SEPHAROSE as described by Deutsch, D. G. and E. T. Mertz,
"Plasminogen: purification from human plasma by affinity
chromatography," Science 170(962):1095-6 (1970).
[0037] SEPHAROSE is a trade name of Pharmacia, Inc. of New Jersey
for a high molecular weight substance for the separation by gel
filtration of macromolecules. The process may be performed on any
plasma source, recombinant source, cell culture source or
transgenic source. For example, plasma from a waste fraction
derived from the purification of immunoglobulin from a
chromatographic process can be used as described in commonly owned
U.S. patent application Ser. No. 09/448,771, filed Nov. 24, 1999,
which is incorporated by reference herein.
[0038] Plasminogen was extracted from this waste fraction (referred
to herein as the "caprylate cake I" (CCI)) over a wide range of pH.
Conditions of extraction can be varied from a pH of about 3.5 to
about 10.5 using a variety of buffers capable of providing a pH in
this range, including citrate, acetate, tris, imidazole, histadine,
HEPES and/or phosphate buffers. The extraction can occur at
temperatures from about 4.degree. C. to 37.degree. C. and can be
run for 1 to 24 hours without deleterious effect. In addition, the
ionic strength can be varied by the addition of about 0.2 Molar
sodium chloride without deleterious effect on the extraction of
plasminogen.
[0039] Following the extraction of plasminogen, lipid and protein
impurities and TSE were reduced by precipitation with the addition
PEG, in a range of about 1 to about 10% weight/volume or the
addition of about 80 to about 120 g/L ammonium sulfate. The PEG or
ammonium sulfate precipitate can be removed by depth filtration.
The resulting solution is then placed on a lysine affinity resin
column.
[0040] Removal of lipid and protein impurities above can be further
enhanced by the addition of a particulate metal oxide. The metal
oxide can be silicon dioxide or aluminum hydroxide. The metal oxide
can also be fumed alumina. The silicon dioxide can be a fumed
silica. The fumed silica can be a fumed silica filter-aid such as
CAB-O-SIL.RTM. M-5P fumed silica from Cabot Corporation, Tuscola,
Ill. (an amorphous, collodial silicon dioxide). Use of a
particulate metal oxide can result in a significant further
reduction in lipids and proteinaceous contaminants such as TSE
pathogenic prion proteins. Use of a fumed silica filter aid, e.g.
CAB-O-SIL, has been shown to result in a further reduction of prion
proteins of from about 2 to about 3 logs, in addition to the
clearance effect of PEG. See FIG. 12 and Example 14 below.
[0041] If desired, the solubility of plasminogen may be enhanced by
the addition of excipients, e.g., omega-amino acids (lysine,
polylysine, arginine, tranexamic acid, or epsilon amino caproic
acid, or combinations or analogues thereof). Solubility enhancement
may be accomplished with from about 0.02 M to about 1 M of a
suitable excipient. Preferably about 0.2 M lysine is sufficient. If
added, the lysine is preferably removed by diafiltration (after the
PEG, fumed silica (e.g. CABOSIL), cation-exchange column
chromatography, and/or ammonium sulfate precipitation and depth
filtration), and the resulting solution placed on a lysine affinity
resin column. The phrase "lysine affinity resin" is used generally
for affinity resins containing lysine or its derivatives or epsilon
caproic acids as the ligand. The column can be eluted with a low pH
solution of approximately 1 to 4.
[0042] The protein obtained after elution from the affinity column
is generally at least 80% plasminogen. The purified plasminogen is
then stored at low pH in the presence of simple buffers such as
glycine and lysine or omega-amino acids. Storage at low pH also
provides an opportunity for viral inactivation and removal and TSE
removal as determined by spiking methods. The studies of the
present invention suggest that plasmin meets the most stringent
requirements for 6 log clearance of non-enveloped viruses including
one 4 log removal step, and 10 log clearance for enveloped viruses
including two orthogonal 4 log elimination steps. In addition to
sufficient virus clearance, the plasmin process of the invention is
characterized by greater than 6 logs of TSE infectivity removal for
added safety.
[0043] The plasminogen in solution is then activated to plasmin by
the addition of a plasminogen activator, which may be accomplished
in a number of ways including but not limited to streptokinase,
urokinase, or the use of urokinase immobilized on resin and use of
streptokinase immobilized on resin. The preferred plasminogen
activator is soluble streptokinase. The addition of stabilizers or
excipients such as glycerol, omega-amino acids such as lysine,
polylysine, arginine, epsilon amino caproic acid and tranexamic
acid, and salt enhance the yield of plasmin.
[0044] Purifying Plasmin
[0045] Plasmin was purified from unactivated plasminogen by
affinity chromatography on resin with benzamidine as the ligand and
eluted with a neutral pH excipient solution or low pH solution.
This step can remove essentially all degraded plasmin as well as
the majority of the streptokinase.
[0046] As a polishing step for the removal of remaining
streptokinase, hydrophobic interaction chromatography (HIC) at low
pH is performed. Following the HIC step, the plasmin is formulated
as a sterile protein solution by ultrafiltration and diafiltration
and 0.22 .mu.m filtration.
[0047] The present method additionally includes the steps of
activating plasminogen to plasmin using a plasminogen activator and
then capturing the formed active plasmin on an active
plasmin-specific absorbent material. The bound plasmin is then
eluted with a low pH buffer. The eluted plasmin is buffered with a
low pH, low buffering capacity agent such as an acid. Typically,
the eluted plasmin is buffered to a pH of between about 2.5 to
about 4.
[0048] The low buffering capacity of the acidic buffer allows the
reversibly inactivated acidified plasmin to be brought up to
physiological pH quickly, becoming activated thereby when
administered as a thrombolytic agent. Typically, the buffer is
added in a concentration at which the pH of the acidified plasmin
is raised to neutral pH by adding serum in an amount no more than
about five times the volume of the acidified plasmin.
[0049] Cleaving the Plasminogen to Yield an Active Plasmin
[0050] Plasminogen can be cleaved to plasmin by using a catalytic
concentration of an immobilized or soluble plasminogen activator.
Plasmin, the principle fibrinolytic enzyme in mammals, is a serine
protease with trypsin-like specificity that is derived from the
inactive zymogen precursor plasminogen circulating in plasma.
Plasminogen itself is a 790 amino acid polypeptide having an
N-terminus glutamate residue. Plasminogen activators such as
soluble streptokinase, tissue plasminogen activator (tPA) or
urokinase will cleave the single-chain plasminogen molecule to
produce active plasmin at the Arg560-Val561 peptide bond. The
resulting two polypeptide chains of plasmin are held together by
two interchain disulfide bridges. The light chain of 25 kDa carries
the catalytic center and is homologous to trypsin and other serine
proteases. The heavy chain (60 kDa) consists of five triple-loop
kringle structures with highly similar amino acid sequences. Some
of these kringles contain so-called lysine-binding sites that are
responsible for plasminogen and plasmin interaction with fibrin,
.alpha.2-antiplasmin or other proteins.
[0051] The activation of plasminogen can occur at about 4.degree.
C. to about 37.degree. C. and typically takes between about 2 to 24
hours. The plasminogen can be cleaved in the presence of
stabilizers or excipients such as omega-amino acids, salts, and
glycerol. The omega-amino acids can include lysine, epsilon amino
caproic acid, tranexamic acid, poly lysine, arginine and
combinations or analogues thereof. Upon the completion of the
activation, the plasmin solution can be filtered and further
stabilized for several days at neutral pH by the addition of
excipients such as omega-amino acids and sodium chloride and
applied to benzamidine-SEPHAROSE.
[0052] Removing Plasminogen Activator and Impurities
[0053] The active plasmin formed from the cleaving of the
plasminogen can then be bound to an active plasmin specific
absorbent to substantially remove the plasminogen activator.
Because the protein of interest is an active serine protease with
trypsin-like specificity, benzamidine may be used as an active
plasmin specific absorbent that allows for the capture of the
active plasmin. Other active plasmin specific absorbents having
similar properties as benzamidine may also be used. The benzamidine
can be immobilized in a solid support medium. The solid support
medium can be a resin or SEPHAROSE. Additionally, hydrophobic
interaction may be used to further remove the plasminogen activator
(see below, Removal of Streptokinase by Hydrophobic Interaction
Resin Chromatography).
[0054] More specifically, the cleaved plasminogen is typically
contained in a solution of amino acids, sodium chloride and
glycerol, which allows for stability of the solution for several
days at neutral pH before it is applied to a benzamidine-SEPHAROSE
column equilibrated with about 0.05 M Tris, pH 8.5, 0.5 M NaCl. The
column is typically run at 4.degree. C. The front portion of the
non-bound peak contains high-molecular weight impurities, with the
rest of the non-bound peak being represented by residual
non-activated plasminogen and by inactive autodegradation products
of plasmin.
[0055] The bound plasmin can then be eluted with an acid buffer or
with a substantially neutral pH excipient solution. The plasmin
bound to benzamidine-SEPHAROSE can be eluted with an acidic buffer
such as glycine buffer. When a substantially neutral pH excipient
solution is used to elute the bound plasmin, the final eluted
plasmin solution can be substantially free of degraded plasmin.
Typically, the substantially neutral pH excipient solution has a pH
of value of between about 6.5 to about 8.5. However, the pH of the
solution can range from about 2.5 to about 9.0. In particular
embodiments, the pH can be from about 4.0 to about 7.5. In other
embodiments, the pH can be about 6.0. Examples of excipients
include omega-amino acids, including lysine, epsilon amino caproic
acid, tranexamic acid, polylysine, arginine, and analogues and
combinations thereof, and salts such as sodium chloride.
[0056] An appropriate concentration of salt can be represented by a
conductivity from about 5 mS to about 100 mS. Generally, the salt
concentration can be varied somewhat inversely in relation to
acidity, i.e. lower pH solutions can work well with lower salt and
solutions having higher pH (within the ranges discussed above) can
work well with higher salt concentrations. When the salt is sodium
chloride, the concentration can be from about 50 mM to about 1000
mM, or from about 100 mM to about 200 mM. When the solution is at
about pH 6.0, the concentration of sodium chloride can be about 150
mM.
[0057] Removal of Streptokinase by Hydrophobic Interaction Resin
Chromatography
[0058] As noted above, the streptokinase activator may be further
removed from plasmin by hydrophobic interaction chromatography. In
particular embodiments, the activated plasmin solution is made
about 0.1 M in ammonium sulfate and subjected to hydrophobic
interaction chromatography, e.g. in a column format using a resin
such as octyl-SEPHAROSE.
[0059] Nanofiltration of Plasmin
[0060] The octyl-SEPHAROSE flow-through containing active plasmin
can be subjected to nanofiltration. The flow-through is generally
subjected to pre-filtration with a 0.1 micron filter capsule, and
then subjected to nanofiltration, e.g. using an ASAHI NF (normal
flow) 1.0 m.sup.2 15N membrane (PLANOVA filters, Asahi Kasei
America, Inc., Buffalo Grove, Ill.). Implementing nanofiltration
further downstream in the process, after octyl hydrophobic
interaction chromatography, improves throughput and membrane flux
properties due to a more pure feedstream.
[0061] Buffering the Plasmin Solution with a Low pH, Low Buffering
Capacity Agent
[0062] The eluted plasmin can be buffered with a low pH, low
buffering capacity agent. The low pH, low buffering capacity agent
typically comprises a buffer of either an amino acid, a derivative
of at least one amino acid, an oligopeptide which includes at least
one amino acid, or a combination of the above. Additionally the low
pH, low buffering capacity agent can comprise a buffer selected
from acetic acid, citric acid, hydrochloric acid, carboxcylic acid,
lactic acid, malic acid, tartaric acid, benzoic acid, serine,
threonine, methionine, glutamine, alanine, glycine, isoleucine,
valine, alanine, aspartic acid, derivatives or combinations
thereof. The buffer can be present in the reversibly inactive
acidified plasmin at a concentration such that the pH of the
acidified plasmin can be raised to neutral pH by adding serum to
the composition in an amount no more than about 4 to 5 times the
volume of acidified plasmin.
[0063] The concentration of plasmin in the buffered solution can
range from about 0.01 mg/ml to about 50 mg/ml of the total
solution. The concentration of the buffer can range from about 1 nM
to about 50 mM. Of course, these ranges may be broadened or
narrowed depending upon the buffer chosen, or upon the addition of
other ingredients such as additives or stabilizing agents. The
amount of buffer added is typically that which will bring the
reversibly inactive acidified plasmin solution to have a pH between
about 2.5 to about 4.
[0064] Further Stabilizing the Inactive Acidified Plasmin
Solution
[0065] The reversibly inactive acidified plasmin solution may be
further stabilized by the addition of a stabilizing agent such as a
polyhydric alcohol, pharmaceutically acceptable carbohydrates,
salts, glucosamine, thiamine, niacinamide, or combinations thereof.
The stabilizing salts can be selected from the group consisting of
sodium chloride, potassium chloride, magnesium chloride, calcium
chloride and combinations thereof. Sugars or sugar alcohols may
also be added, such as glucose, maltose, mannitol, sorbitol,
sucrose, lactose, trehalose, and combinations thereof.
[0066] Concentrations of carbohydrate added to stabilize the
reversibly inactive acidified plasmin solution include a range from
about 0.2% w/v to about 20% w/v. Ranges for a salt, glucosamine,
thiamine, niacinamide and their combinations can range from about
0.01 M to about 1 M.
[0067] Plasmin formulated formulated according to the invention in
buffered acidified water has been found to be extremely stable. It
can be kept in this form for months without substantial loss of
activity or the appearance of degradation products of a proteolytic
or acidic nature. At 4.degree. C., plasmin is stable for at least
nine months. Even at room temperature, plasmin is stable for at
least two months. Long-term stability at room temperature can allow
this formulation to be compatible with long regimens of
thrombolytic administration. For example, 36 hours administration
of thrombolytics such as tissue plasminogen activator or urokinase
is common in treatment of peripheral arterial occlusions.
[0068] The ability of a buffered acidified plasmin to become fully
active upon transfer to physiological pH is evidenced by its
activity in the caseinolytic assay and also in the
I.sup.125-fibrin-labelled clot lysis assays. Both of these assays
are performed at pH 7.4, and there was complete recovery of plasmin
activity during the change of pH and passing through the iso-pI
point (pH 5-5.5). This is because plasmin is formulated in a
non-buffered solvent and when added to a buffered solution (either
PBS or plasma) it adopts the neutral pH instantly and the
precipitation that usually accompanies the slow passage through the
iso-pI point, does not occur.
[0069] A feature of the active plasmin as used in the present
invention is the maintenance of the plasmin in an acidic buffer and
its formulation in acidified water, providing a pure and stable
active plasmin. Its efficacy was demonstrated in in vitro assays
and in an in vivo rabbit jugular vein thrombolysis model unified,
substantially purified or partially purified enzyme such as, but
not limited to, plasmin or any composition containing plasmin that
is within the scope of the present invention.
[0070] A description of a method of treating thrombolysis and
related ailments employing aspects of the claimed invention is
disclosed in the application entitled "Method of Thrombolysis by
Local Delivery of Reversibly Inactivated Acidified Plasmin," U.S.
patent application Ser. No. 10/143,157, commonly assigned, and
incorporated herein by reference in its entirety. Additionally,
compositions made in accordance with the claimed invention are
disclosed in the application entitled "Reversibly Inactivated
Acidified Plasmin," U.S. patent application Ser. No. 10/143,112,
and commonly assigned, and incorporated herein by reference in its
entirety.
[0071] The following examples are given only to illustrate the
present process and are not given to limit the invention. One
skilled in the art will appreciate that the examples given only
illustrate that which is claimed and that the present process is
only limited in scope by the appended claims.
EXAMPLES
Example 1
[0072] Caprylate Cake I (CCI) Extraction and Lipid Reduction by PEG
Precipitation and Filtration
[0073] Caprylate cake I (CCI) is a fraction resulting from a pH 5
caprylate precipitation of resuspended Cohn Fractions II+III in the
IGIV-C process (see, e.g., Lebing, W. et al. Vox Sang,
84(3):193-201 (April 2003)). Plasminogen (Pmg) is extracted from
the CCI by solubilizing at a cake:buffer ratio of about 1:10 for 2
to 3 hours at 4.degree. C. with mixing. While several extraction
solutions were investigated, the current method was performed with
100 mM Tris pH 10.5 to maintain the pH at or above neutral; a
condition favorable to Pmg solubilization from the CCI. Table 1
depicts the extraction solutions investigated along with their
final extract pH and Pmg potency.
1TABLE 1 CCI Extraction Solutions: Resulting Final Extract pHs and
Pmg Activities. Extraction Solution Final Extract pH Pmg (IU/ml)
0.1 M Tris pH 10.5 9.2-9.5 1.77 0.2 M Tris pH 7.5 7.5 2.06 0.05 M
Citrate, 0.2 M .epsilon.-ACA, 0.4 M 6.0 1.49 NaCl pH 6.5 0.15 M
Citrate pH 8.3 6.7 1.21 0.4% Acetic Acid pH 3.5 3.5 0.05
[0074] Following 2 to 3 hours of extraction, the temperature of the
extract is adjusted to 20.degree. C. and the pH to 7.5. Table 2
shows the Pmg yield, based on nephelometry, from Clarified Plasma
Pool through Fraction II+III and CCI Extract.
2TABLE 2 Step and Process Yields for Pmg from Clarified Plasma Pool
to CCI Extract. % Pmg Step % Pmg Process Cohn Fraction mg Pmg/g
(SD), n Yield Yield Clarified Plasma 0.124 (0.013), 33 Pool
Fraction II + III 0.143 (0.024), 30 65.6 CCI Extract 0.145 (0.01),
7 101 66.3 (post L-lysine)
[0075] Only about 66% of the Pmg in plasma tracks to Fraction
II+III while virtually all of the Pmg found in the resuspended
Fraction II+III precipitates to and is extracted from CCI.
Extraction of CCI in Tris pH 10.5, final CCI Extract pH of 9.2-9.5,
solubilizes all of the Pmg found in the CCI.
[0076] The addition of lysine derivatives (100 mM L-lysine, 50 mM
epsilon amino caproic acid (EACA)) increases the solubility of Pmg
in the CCI Extract resulting in increased recoveries during
subsequent PEG precipitation and filtration steps as illustrated in
FIG. 1.
[0077] Reduction of lipid is achieved through precipitation by the
addition of PEG 3350 to 3%-4% w/w. As mentioned previously, the
addition of L-lysine to 100 mM prior to PEG addition is necessary
to maintain high Pmg recovery in the PEG filtrate, or about 90%.
Without the addition of lysine, only about 25% of the Pmg is
recovered in the PEG filtrate (FIG. 1). The PEG precipitation
proceeds for 1 to 2 hours at 20.degree. C. with mixing. Filter aid
is added to 4% w/w and mixed prior to depth filtration through a
CUNO 30SP followed by further clarification with 0.5 micron and
0.22 micron filters.
[0078] FIG. 1 shows the lipid content, determined by cholesterol
and triglycerides concentration, is reduced by 60-70% following PEG
precipitation and filtration (CCI Filtrate I). The CCI Filtrate I
is diluted 1:1 with phosphate buffered saline pH 7.5 and held at
20.degree. C. for 1 to 2 hours as precipitation often continues
following filtration. The CCI Filtrate I is filtered through 0.5
.mu.m and 0.22 .mu.m filters to remove any additional precipitate;
CCI Filtrate II. Nephelometry data for CCI Extract and CCI
Filtrates I and II are illustrated in FIG. 2. Note that fibrinogen
and apolipoprotein A-1 concentrations are reduced following PEG
precipitation.
[0079] The CCI Filtrate II is diafiltered by tangential flow
filtration (TFF) against phosphate buffered saline pH 7.5 to reduce
the L-lysine concentration such that it will not act as a
competitive inhibitor for Pmg binding to the lysine affinity resin.
Experiments were performed to illustrate the necessity of lysine
removal. Loading the CCI Filtrate II directly onto a lysine
affinity resin without reduction in soluble lysine concentration,
results in the capture and release of about 4% of the Pmg activity.
Diluting the CCI Filtrate II 1:1 with TBS (10 mM Tris, 150 mM NaCl
pH 7.5) still resulted in capture and release of only about 5% of
the Pmg activity. Following 5 volumes of diafiltration to reduce
the lysine concentration, about 22% of the Pmg activity was
captured and released from the lysine affinity resin (in
retrospect, the column was overloaded by about 50%).
[0080] Constant volume diafiltration was performed by tangential
flow filtration (TEF) against 5 volumes phosphate buffered saline
pH 7.5 using a 30 kDa molecular weight cutoff membrane. Following
diafiltration, the protein solution was concentrated by
ultrafiltration to 4 to 5 A.sub.280/ml. Pmg recoveries in the UF/DF
retentate, by nephelometry, averaged 84% (.+-.1, n=3). FIG. 3 shows
reduced SDS PAGE for each of the process intermediates discussed
thus far. The data in FIGS. 2 and 3 illustrate the complexity and
heterogeneity of the CCI Extract and subsequent Filtrates.
Example 2
[0081] Purification of Pmg by Lysine Affinity Chromatography:
[0082] The purpose of lysine affinity chromatography is to purify
Pmg, which represents from about 3 to 5% of the total protein in
the diafiltered CCI Filtrate II. The DF CCI Filtrate II was applied
to a Lysine-SEPHAROSE 4B (Amersham Pharmacia #17-0690-01) column
equilibrated with 0.01 M NaH.sub.2PO.sub.4, 0.15 M NaCl pH 7.5, at
3.5-4.0 A.sub.280/ml resin. Unbound proteins were washed through
the column with the equilibration buffer and the resin was then
washed with 0.01 M NaH.sub.2PO.sub.4, 0.5 M NaCl pH 7.5 to remove
non-specifically bound protein; no protein was removed. Bound
protein, Pmg, was eluted with 0.1 M Glycine, 0.03 M Lysine pH 3.0
and collected with mixing to maintain low pH. FIGS. 4 and 5 show
SDS PAGE analysis and the chromatogram of the lysine affinity
purification of Pmg, respectively. The resin was cleaned
sequentially with 0.1 N NaOH and 2.0 M NaCl, 0.1% Triton X-100 and
stored in 20% ethanol. Table 3 shows Pmg step yield by nephelometry
and purity by reduced SDS PAGE.
3TABLE 3 Lysine Affinity Chromatography Pmg Step Yield and Purity
Process Intermediate Step Yield % Pmg Purity % Lysine-SEPHAROSE 4B
Eluate 75.7 85.9
Example 3
[0083] Viral Inactivation and Removal and TSE Removal
[0084] Nanofiltration
[0085] The optimal placement of a nanofiltration step during the
Plasmin process, along with determining the optimal conditions for
pathogens removal from Pmg lysine affinity eluate (Pmg) for a
particular nanofiltration scheme was tested. Pmg was spiked with
porcine parvovirus (PPV) or bovine diarrhea virus (BVDV) and
filtered through a PALL DV20 filter membrane. All runs were
performed with 50 ml starting material (0.3 mg/ml Pmg), 30 psi
constant pressure, pH 3.4 and room temperature. The challenge
solution was pre-filtered through 0.22 .mu.m prior to
nanofiltration. The determining factors for the optimal conditions
for removal of different pathogens by nanofiltration deal mainly
with the attainment of a minimum of 4 log infectivity removal of
known pathogens, percent product recovery, percent potency
remaining, product concentration and product pH. It was found that
PPV and BVDV clearance was >4 log.sub.10 TCID.sub.50. The
nanofiltration step has also the capability of removing greater
than 4 log of TSE. All product recoveries obtained in the study
were .gtoreq.95% with no substantial change in Pmg activity.
[0086] Caprylate Viral Inactivation.
[0087] Because caprylate inactivation is very much pH dependent and
more efficacious under acidic pH conditions, virus inactivation by
caprylate at the low pH lysine affinity chromatography elution step
was examined. BVDV was used as a model enveloped virus to study
caprylate virucidal activity in lysine affinity eluate. Complete
BVDV inactivation, resulting in .gtoreq.4.4 log.sub.10 reduction,
was detected at the lysine affinity column eluate with 3 mM
caprylate at pH 3.4 during 30 min of incubation at room temperature
in the presence of 1.5 mg/ml Pmg. In the absence of product,
complete BVDV inactivation (.gtoreq.4.7 log.sub.10 reduction) was
also achieved with 3 mM caprylate after 30 minutes at pH 3.4. No
visible precipitation was observed during the caprylate treatment
suggesting that the product and virus spike remain soluble and are
not being precipitated by the caprylate. The impact of the added
caprylate on product recovery or potency following lysine affinity
column chromatography was minimal.
[0088] PEG Precipitation
[0089] The effect of PEG on TSE removal was investigated. The
clarification and removal of lipids achieved by depth filtration
and 3% PEG precipitation of the Caprylate Cake I Extract resulted
in greater than 2 log.sub.10 of TSE removal.
4TABLE 4 Total Virus/TSE clearance across Plasmin process Step BVDV
PPV TSE Nanofiltration >4 log 4 log 4 log 3 mM Caprylate >4
log <1 log <1 log Lysine Affinity 3.3 log 2.5 log pending PEG
precipitation <1 <1 2-3 logs Total clearance >12 >6
>6
Example 4
[0090] Streptokinase (SK) Activation of Pmg to Pm (Pm):
[0091] The addition of SK to the purified Pmg solution effects the
conversion of Pmg to Pm. The lysine affinity column eluate pH 3.4
is concentrated by TFF to 2 mg/ml through a 30 kD molecular weight
cutoff membrane. The Pmg solution temperature is ramped down to
4.degree. C. and a Pmg stabilizer, EACA, is added to a final
concentration of 20 mM to protect Pmg against damage during pH
adjustment from 3.4 to 7.5. Without the addition of EACA, a 67 kDa
species appears following the pH swing. The presence of EACA during
pH adjustment results in decreased Pmg degradation as compared to
pH adjustment without EACA (FIG. 6). Once the pH is adjusted to
7.5, the Pmg solution is diluted 1:1 with 20% glycerol, 4.degree.
C., to achieve a final condition of 1 mg Pmg/ml 0.05 M glycine,
0.015 M L-lysine, 0.01 M EACA, 10% glycerol pH 7.5. These
conditions have been optimized for minimizing Pm autodegradation.
SK is added to this solution at a 100:1 Pmg:SK molar ratio. The SK
reaction mixture is mixed at 4.degree. C. for 16 hours to allow
activation of Pmg to Pm. The average relative percent purity, as
determined by reduced SDS PAGE, of each of 4 groups of protein
species (Pmg, Pm HC, Pm LC and impurities/clipped Pm) from 14 SK
activation reactions are listed in Table 5.
5TABLE 5 Relative Average % of Pmg, Pm (HC, LC) and
Impurities/Clipped Pm by Reduced SDS PAGE Following SK Activation;
n = 14. Protein Average % Purity SD Pmg 20.3 5.3 Pm 68.5 4.4 Pm
Heavy Chain 49.0 2.9 Pm Light Chain 19.4 1.5 Impurities/Clipped Pm
11.3 1.8
[0092] The data shows that the SK activation is reproducible and
results in only about 11% clipped Pm/impurities while activation of
Pmg to Pm is about 80%. To stop the activation and Pm
autodegradation reactions, NaCl and EACA are added to final
concentrations of 0.5 M and 0.25 M, respectively. This solution is
stable with respect to Pm integrity, for at least 4 days at
4.degree. C. FIG. 7 illustrates that there is no change in the Pm
purity or Pm autodegradation (Other) over this time period.
Example 5
[0093] Purification of Pm by Benzamidine Affinity
Chromatography:
[0094] The purpose of benzamidine affinity purification is the
separation of unactivated Pmg and impurities, including Pm
degradation products, from active Pm. The stable SK activation
solution, pH adjusted to 8.5 in 0.05 M glycine, 0.015 M L-lysine,
0.25 M EACA, 0.5 M NaCl, 10% glycerol, is applied to a
Benzamidine-SEPHAROSE 6B (Amersham Pharmacia #17-0568-01) column
equilibrated with 50 mM Tris, 500 mM NaCl, pH 8.5. The Pm, both
clipped and intact, is captured by the affinity resin while the
aforementioned impurities flow through the column. The column is
washed with the equilibration buffer until the absorbance at 280 nm
reaches baseline. The bound Pm is then eluted in either one of two
ways: 1) removing the resin and eluting in batch format with 0.1 M
Glycine, 0.03 M Lysine pH 3.4; 2) eluting in a column format with 1
M EACA pH 7.5. Elution with EACA pH 7.5 removes only the intact Pm
while damaged Pm remains bound to the resin. FIG. 8 shows a typical
column format EACA elution profile, including a low pH EACA step to
strip all remaining protein. Elution buffer excipient concentration
(0.25 to 1.0 M EACA), salt concentration (0.1 to 1.0 M NaCL), and
pH (5.0-7.5) conditions can be adjusted to accomplish the goal of
purifying intact Pm.
[0095] The batch elution profile consists only of the unbound
protein peak as the resin is then removed from the column for Pm
elution. The Pm captured and eluted from the affinity resin is
87-91% intact (non-autodegraded) as illustrated in FIG. 9 and
.gtoreq.99% total Pm. The elution of Pm from the benzamidine resin
with EACA was unexpected as lysine derivatives such as EACA
interact with the heavy chain of Pm while benzamidine interacts
with the light chain.
Example 6
[0096] Removal of the Pmg Activator SK
[0097] The purpose of these steps is to remove the Pmg activator SK
such that the only remaining fibrin clot dissolution activity is
that of Pm. The benzamidine affinity step removes >99% of the SK
from the Pm as is illustrated in Table 6.
6TABLE 6 SK removal, as determined by ELISA, by benzamidine
affinity chromatography and hydrophobic interaction chromatography.
Plasmin Process Step Streptokinase (ng/ml) SK activation 1930.1
Benzamidine-SEPHAROSE unbound 1549.5 Benzamidine-SEPHAROSE eluted
Pm 1.9 HIC Unbound Pm 0.7 HIC NaOH strip (SK) 1.3 Final Formulation
Pm <0.5
[0098] The hydrophobic interaction step using Octyl SEPHAROSE 4 FF
(Amersham Pharmacia #17-0946-02) acts as a polishing step to remove
essentially any remaining SK. The final sterile Pm product has no
detectable SK by ELISA. The 1 M EACA eluate pH 7.5, from the
benzamidine affinity column, is adjusted to pH 3.4 and
(NH.sub.4).sub.2SO.sub.4 is added to a final concentration of 0.1
M. This acts as the protein load for the Octyl-SEPHAROSE 4 FF
column. The column is equilibrated with 0.1 M
(NH.sub.4).sub.2SO.sub.4, 0.1 M Glycine, 30 mM Lysine pH 3.4. Pm
flows through the column while SK binds to the column and is
separated from Pm. The captured SK is removed from the resin along
with 0.1 to 1.0 N NaOH. FIG. 9 is an Octyl-SEPHAROSE 4 FF
chromatogram from a proof of principle experiment. Pmg and SK were
mixed at a 2:1 Pmg:SK molar ratio and subjected to Octyl-SEPHAROSE
4 FF chromatography. The high levels of SK were used so it could be
tracked throughout the chromatographic cycle using an anti-SK
western blot. FIG. 10 illustrates the removal of SK from the Pm by
SDS PAGE and anti-SK western blot. The SK standard (panels A and B;
lane 1) migrates true to its molecular weight of 47 kDa. Once mixed
with Pmg, the SK is modified and migrates faster and as several
species. There is no detectable SK in the unbound protein fraction,
which contains the bulk of the Pm, by anti-SK western blot (panel
B; lane 3).
[0099] Results for final sterile preparations of Pm purified by
benzamidine affinity and HIC chromatographies, as described above,
are listed in Table 7.
7TABLE 7 Relative Average % Purity of Pm (HC, LC) by Reduced SDS
PAGE Following HIC; n = 2. Protein Average % Purity Pmg 0.0 Pm 95.5
Pm Heavy Chain 66.5 Pm Light Chain 29.0 Impurities/Clipped Pm
4.5
[0100] Examples 7 through 15 below show additional embodiments of
the process of the invention for preparation of plasmin from the
Caprylate Cake I starting material.
Example 7
[0101] Caprylate Cake I (CCI) Extraction of Plasminogen
[0102] Caprylate Cake I (CCI) is suspended in 10 volumes (w/w) of
pH 8.0, 0.05 M phosphate buffer containing 0.2 M lysine, 0.25%
(w/w) CAB-O-SIL M-5P fumed silica (Cabot Corp. Tuscola, Ill.), and
3.5% (w/w) PEG 3350. These components are mixed at ambient
temperature until the CCI becomes a homogeneous suspension by
visual examination (not less than 4 hours). During this time, the
pH is checked hourly, and if the pH drops below 7.30, 1.0 N NaOH is
added to adjust the pH to 7.30-7.60 (target pH 7.50) (the pH drops
during extraction due to the low pH (5.0) of the CCI).
[0103] After suspension is complete, 1% (w/w) of CELPURE P1000
filter aid (Sigma-Aldrich Co., St. Louis, Mo.) is added and mixed
until evenly dispersed. The suspension is then filtered using CUNO
90 SP filter pads (Cuno, Inc., Meriden, Conn.) using press
filtration (target 20 psi). Prior to filtration, the press and
filters are rinsed with cold water for injection (CWFI). The filter
is rinsed with 1.5 cake volumes (w/w) of rinse buffer pH 7.3, 0.05
M phosphate buffer containing 0.2 M lysine, and 3.5% (w/w) PEG
3350.
[0104] The press filtrate is cooled to between 10.degree. C. and
14.degree. C. (target 12.degree. C.) and 3 M NaCl is added to a
final concentration of 0.5 M. The solution is then concentrated to
a target of 58% of starting volume by ultrafiltration using a 30 kD
polyethersulfone (BIOMAX) PELLICON 2 membrane cassette (Millipore
Corporation, Billerica, Mass.). Prior to use, the ultrafiltration
system is flushed with WFI until the permeate is between pH 5.0 and
7.0, followed by pre-conditioning with 0.01 M sodium phosphate, 0.5
M NaCl, pH 7.5. During filtration, the temperature is maintained
between 10.degree. C. and 14.degree. C.
[0105] The concentrated solution is then subjected to diafiltration
with not less than 5 volumes of 0.01 M sodium phosphate, 0.5 NaCl,
pH 7.5. The solution is maintained between 10.degree. C. and
14.degree. C. When diafiltration is complete, the retentate valve
is opened, the permeate valve is closed, and the membrane is swept
at maximum retentate flow for 15 to 20 minutes. Using process air,
the remaining product is blown out from the ultrafiltration
skid/cassettes into the filtrate tank for no more than 2 minutes at
9 to 11 psi.
[0106] The diafiltrate is then subjected to ECH lysine-SEPHAROSE
4FF (Amersham Biosciences Corp., Piscataway, N.J.) affinity
chromatography for the purification of plasminogen. The
pre-equilibration buffer is 0.05 M sodium phosphate, pH 7.5; the
equilibration buffer is 0.01 M sodium phosphate, 0.5 NaCi, pH 7.5;
and the elution buffer is 0.1 M glycine, 0.03 M L-lysine (HCl), pH
3.0. The entire chromatographic system (buffers, column, bioprocess
skid) are allowed to equilibrate to a temperature between 2.degree.
C. to 8.degree. C. A MILLIPORE POLYGUARD 0.3 .mu.m filter is placed
in-line for running buffers. The diafiltrate is filtered with an
OPTICAP 0.2 .mu.m nominal filter (Millipore Corp.) or its
equivalent prior to chromatography.
[0107] The column is pre-equilibrated with 4 column volumes of
pre-equilibration buffer. The column is then equilibrated with
equilibration buffer until the effluent pH is stabilized at 7.4 to
7.6 and the conductivity is stable at 38 to 48 mS. The diafiltrate
is then loaded onto the column while the temperature is maintained
at between 2.degree. C. and 8.degree. C. The column is washed with
4 volumes of equilibration buffer. The column is eluted with lysine
elution buffer and plasminogen is collected when the pH slope is
-0.5. Collection is terminated when the UV absorbancy of the eluate
peak is no more than 0.1 AU (absorbance units). All buffers,
diafiltrate load, and washes are run in the downward direction at a
flow rate of 100 cm/hr.
[0108] An alternative to ultrafiltration/diafiltration (UF/DF) for
removal of lysine is cation-exchange (CIEX) column chromatography.
Using a resin with a high ionic capacity and low pore retention
(e.g., Dowex 50Wx8 100-200 mesh; Dow Chemcals) it is possible to
bind only small molecules like lysine, while proteins remain
unbound in the flowthrough fraction. The CIEX, and then the lysine
column are equilibrated with 0.05 M sodium phosphate, pH 7.0 to
7.5, and operated throughout at chilled or ambient temperature
(2.degree. C. to 22.degree. C.). The CUNO filtrate is filtered with
an OPTICAP 0.2 .mu.m nominal filter (Millipore Corp.) or its
equivalent prior to chromatography, then applied onto the CIEX
column at 50 cm/h. The unbound protein in the CIEX column
flowthrough are then applied directly to the lysine affinity
column, connected in series, to purify the plasminogen. The lysine
column is eluted with lysine elution buffer and plasminogen is
collected when the pH slope is -0.5. Collection is terminated when
the UV absorbancy of the eluate peak is no more than 0.1 AU
(absorbance units). All buffers, CUNO filtrate load, and washes are
run in the downward direction at a flow rate of 100 cm/hr.
[0109] The eluate is frozen at no more than -20.degree. C. for
storage.
Example 8
[0110] Activation of Plasminogen
[0111] Plasminogen prepared according to Example 7 is activated to
plasmin with streptokinase as follows:
[0112] Frozen lysine eluate (plasminogen) is thawed to a target
temperature of 22.degree. C. (20.degree. C. to 24.degree. C.).
Plasminogen is incubated with sodium caprylate for viral
inactivation for no longer than 1 hour, at a final sodium caprylate
concentration of 0.0042 M (0.0034 to 0.0048 M) at a target pH of
3.4 (3.1 to 3.5), with the temperature maintained at the target of
22.degree. C.
[0113] Following caprylate incubation, the plasminogen solution is
diluted to 1.70 A.sub.280 (1.45-1.95 range) using an Activation
Dilution Buffer of 0.1 M glycine, 0.03 M L-lysine, target pH of
3.40 (3.15 to 3.45). Plasminogen is activated to plasmin with
streptokinase at a molar ratio of 100:1, plasminogen to
streptokinase, in 0.010 M EACA, 0.010 M sodium phosphate, pH 7.0
(6.90 to 7.10), at a target temperature of 5.degree. C. (2.degree.
C. to 8.degree. C.), for 8 hours (7.5 to 8.5). The activation is
quenched by addition of EACA and NaCl to a final concentration of
0.25 M EACA and 0.5 M NaCl. The pH is adjusted to a target of 8.50
(8.40 to 8.60) with 1.0 N sodium hydroxide.
[0114] Activated plasmin is purified using benzamidine-SEPHAROSE
4FF (Low Sub) affinity resin (Amersham Biosciences Corp.,
Piscataway, N.J.). The benzamidine-SEPHAROSE resin is poured into a
450.times.500 column. The equilibration (wash) buffer is 0.05 M
Tris-base, 0.5 M NaCl, with a target pH of 8.50 (8.40-8.60).
Elution buffer is 0.25 M EACA, 0.15 M NaCl, with a target pH of
6.00 (5.90-6.10). All buffers and plasmin flow in the downward
direction on the column at a flow rate of 100 cm//hr unless noted
specifically as otherwise. The column is equilibrated with wash
buffer until effluent pH is stable at 8.25 to 8.75 and until
conductivity is stable at 36 to 48 mS. Activated plasmin is then
loaded onto the column while maintaining the temperature between
2.degree. C. and 8.degree. C. The column is washed with no less
than 3 column volumes of wash buffer and the plasmin is eluted with
elution buffer. The eluate is adjusted to a target pH of 3.40
(3.30-3.50) with 1.0 N HCl with mixing at 2.degree. C. to 8.degree.
C.
Example 9
[0115] Removal of Streptokinase
[0116] The benzamidine-SEPHAROSE eluate is further processed for
removal of streptokinase by octyl-SEPHAROSE 4FF hydrophobic
interaction chromatography (resin available from Amersham
Biosciences Corp., Piscataway, N.J.). The resin is poured into a
140.times.500 column, packed, and qualified according to the resin
manufacturer's instructions. The octyl-SEPHAROSE equilibration
(wash) buffer is 0.1 M glycine, 0.03 M L-lysine, 0.1 M ammonium
sulfate, at a target pH of 3.40 (3.30-3.50). A MILLIPORE POLYGUARD
0.3 .mu.m filter is placed in-line when running buffers and loading
sample. Buffers, column, and bioprocess skid are all equilibrated
to between 2.degree. C. and 8.degree. C. prior to use.
[0117] All buffers and sample load are run in a downward direction
at a flow rate of 200 cm/hr unless otherwise specifically noted.
The packed column is equilibrated with wash buffer until the
effluent pH is stable at 3.00 to 3.50 and the conductivity is
stable at 16 mS to 26 mS. After ammonium sulfate is added to the
purified plasmin solution (benzamidine-SEPHAROSE eluate prepared as
in Example 8) to 0.1 M, the plasmin is applied to the resin at a
target pH of 3.40 (3.30-3.50), and at a temperature between
2.degree. C. and 8.degree. C. The plasmin is collected in the
flow-through.
Example 10
[0118] Nanofiltration of Plasmin
[0119] The plasmin solution (the octyl-SEPHAROSE flow-through) from
Example 9 is subjected to nanofiltration using PLANOVA 15N filters
(ASAHI NF 1.0 m.sup.2 membrane, 15N) (Asahi Kasei America, Inc.,
Buffalo Grove, Ill.). Prior to nanofiltration, the octyl-SEPHAROSE
flow-through is subjected to filtration using a MILLIPORE 0.1
micron 4" or 10" OPTICAP filter capsule. A peristaltic pump and
silicon tubing are used for these filtration processes. A leakage
test is performed on the nanofilter prior to use.
[0120] The capacity of the nanofilter is no more than 30 g
plasmin/m.sup.2. An in-line pressure gauge is used for feed during
nanofiltration. The system is rinsed with octyl-SEPHAROSE wash
buffer (see Example 9), and the plasmin-containing flow-through is
pumped through the nanofilter at a target pressure of 12 psi (10
psi -14 psi).
Example 11
[0121] Ultrafiltration/Diafiltration of Plasmin Nanofiltrate
[0122] A peristaltic pump with BIOPRENE tubing (Watson-Marlow
Bredel Inc, Wilmington, Mass.) is used in conjunction with a
PELLICON-2 steel holder and MILLIPORE 10 kD BIOMAX UF cassettes
(Millipore Corporation, Billerica, Mass.). The process temperature
is maintained between 2.degree. C. and 12.degree. C. The
ultrafiltration system is flushed with CWFI until the permeate pH
is between 5.00 and 7.00. The system is then flushed with 0.002 M
acetic acid until the permeate and retentate pHs are between 3.10
and 3.50. The system is cooled to between 2.0.degree. C. and
8.0.degree. C. before product is committed to the system. The
nanofiltrate of Example 10 is then concentrated to a target
A.sub.280 of 5.1 (4.0 to 6.0) by ultrafiltration.
[0123] The concentrated solution is then diafiltered with no less
than 5 volumes of 0.002 M acetic acid, target pH of 3.20
(3.10-3.30) while the temperature is maintained between 2.degree.
C. and 12.degree. C. The diafiltered solution is concentrated to a
target A.sub.280 of 12.0 (11.0-13.0), and the pH is adjusted if
necessary to between 3.10 and 3.30 (target 3.20).
Example 12
[0124] Plasmin Formulation
[0125] The diafiltered plasmin from Example 11 is formulated at 5
mg plasmin per ml of a solution containing 5.1%
trehalose-dihydrate, 2 mM acetic acid, pH 3.1-3.3 (target 3.2). The
plasmin can be bulked with trehalose and then adjusted to a target
potency of 5.25 mg/ml and transferred into STEDIM 4 liter EVA bags
(STEDIM, Inc., Concord, Calif.).
[0126] The plasmin can be optionally frozen at no more than
-50.degree. C. and stored at no more than -20.degree. C.
Example 13
[0127] Effect of CAB-O-SIL M-5P on Plasminogen and Lipid Levels in
PEG/CUNO Filtrate
[0128] Experiments (with 3.0% PEG) showed that the addition of
CAB-O-SIL M-5P to Caprylate Cake I (CCI) suspensions greatly
reduced lipid levels with no loss in plasminogen recovery. To
determine an appropriate CAB-O-SIL M-5P concentration to further
reduce filtrate lipid levels, CCI suspension was treated for three
hours with 3.0% PEG and 0.1%, 0.25%, 0.5% or no CAB-O-SIL M-5P,
followed by depth filtration through CUNO 90SP pads. The PEG/CUNO
filtrates were analyzed for plasminogen (by potency) and lipid
concentrations and the results are shown below.
8TABLE 8 Effect of CAB-O-SIL on Plasminogen and Lipid Levels
CAB-O-SIL M-5P Plasminogen Triglycerides (%) (g/L) Cholesterol
(g/ml) (g/ml) 0.00 0.104 46 <40 (control) 0.10 0.100 20 <40
0.25 0.102 <20 <40 0.50 0.097 <20 <40
[0129] Increasing concentrations of CAB-O-SIL M-5P resulted in
increased lipid clearance without impact on plasminogen recovery.
Based on these findings, a concentration of 0.25% CAB-O-SIL M-5P
was selected as the lowest concentration providing lipid removal to
the level of assay detection.
Example 14
[0130] Effect of CAB-O-SIL M-5P on Pathogenic Prion Protein
Clearance
[0131] Caprylate Cake I (CCI) was suspended in 10 volumes Tris
buffer (pH 7). After 2 hours of mixing, 1% CELPURE P1000 filter aid
was added and mixed for 2 minutes. Crude sheep brain homogenate
(SBH) was added, and an input sample was removed. The remaining
sample was divided into two 100 ml aliquots. One aliquot received
0.25% CAB-O-SIL, the other no CAB-O-SIL. The results are shown in
FIG. 12. A "prove" sample (containing the same SBH innoculate, but
subjected to no processing prior to parallel analysis for prion
protein) showed 5 logs of PrP.sup.Sc. The "No Cab-O-Sil" filtrate
had 3 logs of PrP.sup.Sc. The signals present in the filtrate of
the "0.25% Cab-O-Sil" were not PrP-related and the use of 0.25%
CAB-O-SIL improved the clearance to 3 logs over the "No Cab-O-Sil"
treated sample.
Examples 15
[0132] Use of Aluminum Hydroxide for Pathogenic Prion Protein
Clearance
[0133] Bovine serum albumin (BSA) was dissolved in phosphate
buffered saline (PBS) to create a solution at 1 mg/ml BSA. The BSA
solution was "spiked" with scrapie brain homogenate (SBH; prepared
using hamster brains infected with the 263K hamster-adapted agent),
highly clarified prior to use by centrifugation at 10,000 g for 10
minutes to a final concentration of approximately 1%. CAB-O-SIL
M-5P silica (CAB-O-SIL) was added at various concentrations,
followed by vortexing and filtration using a 0.8 .mu.m filter
(filtration alone was estimated to account for approximately 0.5
log reduction in PrP.sup.Sc). These samples were used to evaluate
aluminum hydroxide (Al.sub.2O.sub.3, 1.9-2.2% (w/v) as a gel or
slurry--represent also as Al(OH).sub.3 or aluminum hydroxide
herein) (ALHYDROGEL, Superfos Biosector A/S, Denmark) as an agent
useful for prion clearance. The volume/volume percentages below and
through refer to the proportion of the ALHYROGEL product added
Various amounts of Al(OH).sub.3 (final concentrations of 0 to 18%
(v/v) as indicated in Table 1) were added to samples containing
SBH, and the samples were mixed. The samples were then centrifuged
at 5100 g for 5 minutes, and the supernatant and pellet were
assayed for PrP.sup.Sc. For the 1% SBH, clearance was greater than
4 logs for aluminum hydroxide when treated with more than 4.5%
(v/v). For 0.1% SBH clearance was greater than 3 logs for aluminum
hydroxide greater than 1% (v/v).
[0134] In order to validate a model system for evaluating
PrP.sup.Sc clearance according to a particular embodiment of the
present invention, a scaled-down model for Caprylate Cake I (CCI)
extraction (as discussed above regarding plasminogen purification
procedure) was characterized with respect to the clearance effect
of the PEG Precipitation/Depth Filtration Steps. The purpose of
this study was to establish a bench-scale model of the CCI
Extraction and PEG Precipitation/Depth Filtration step in the
Plasminogen Process under standard conditions. Once established,
the model system was used to evaluate PrP.sup.Sc clearance across
the process step.
[0135] Briefly, CCI was resuspended in 0.1 M TRIZMA base extraction
buffer (pH 10.5) at 40.degree. C. while mixing for 2-3 hours.
Following extraction, the pH of the solution was adjusted to 7.5
and temperature of the extract increased to 20.degree. C. L-lysine
was added to the extract to a final concentration of 100 mM, while
maintaining a pH of 7.5. Polyethylene glycol (PEG) was added to a
final concentration of 3% (w/w) followed by the addition of HYFLO
SUPERCEL filter aid (Celite Corporation, Lompoc, Calif.) to a final
concentration of 4% (w/w). The extract was then filtered through a
CUNO SP-30 filter pad and filtrate collected. Samples were
collected from initial CCI extract, filtrate, and extract. Total
protein determined by A.sub.280 and plasminogen recovery determined
by immunonephelometry. Recovery analysis indicated very little
protein loss across this step.
[0136] Next, PrP.sup.Sc clearance during the CCI and PEG
precipitation/depth filtration step was evaluated. The purpose of
this experiment was to determine the amount of PrP.sup.Sc removed
during the extraction of the CCI and PEG precipitation/depth
filtration steps. The protocol was the same as described above,
except that during the extraction of CCI, 1 ml of 10% crude SBH was
added into 100 ml of the extract resulting in 0.1% final SBH
concentration. The paste retained by the CUNO SP-30 filter was
resuspended to original volume in TBS. Samples from the Prove
(spiked extract prior to filtration), filtrate, and from the paste
resuspension were analyzed for both plasminogen and PrP.sup.Sc by
Western analysis. The steps above, with no aluminum hydroxide or
CAB-O-SIL, resulted in 1 log of clearance PrP.sup.Sc.
[0137] The effect of 10% (v/v) Al(OH).sub.3 (ALHYDROGEL, Superfos
Biosector A/S, Denmark) on plasminogen recovery and PrP.sup.Sc
clearance during the PEG precipitation/depth filtration was
determined. Protocol was as described above in Example 4, except
that, following the addition of 3% PEG, 10% Al(OH).sub.3 (v/v) was
added. The paste retained by the CUNO SP-30 filter was resuspended
to original volume in TBS. Samples from the Prove (spiked extract
prior to filtration), filtrate, and from the paste resuspension
were analyzed for both plasminogen and PrP.sup.Sc by Western
analysis. Including Al(OH).sub.3 (v/v), as indicated above,
resulted in an increase in PrP.sup.Sc clearance by 2 logs
(approximately 3 logs with versus 1 log without).
[0138] The effect of 3% Al(OH).sub.3 on PrP.sup.Sc clearance during
processing of Caprylate Cake I (CCI) was also determined. CCI was
extracted and processed as described above. In one experiment, both
SBH spike and 3% Al(OH).sub.3 (v/v) were added prior to the cloth
(porous polypropylene filtration. Samples were removed from the
Input (Prove) and cloth filtrate. The presence of PrP.sup.Sc was
determined in each sample by Western analysis. Inclusion of 3%
Al(OH).sub.3 (v/v) resulted in 2 logs of clearance of PrP.sup.Sc.
Without Al(OH)3, clearance was 0 logs.
[0139] While specific embodiments have been set forth as
illustrated and described above, it is recognized that variations
may be made with respect to disclosed embodiments. Therefore, while
the invention has been disclosed in various forms only, it will be
obvious to those skilled in the art that many additions, deletions
and modifications can be made without departing from the spirit and
scope of this invention, and no undue limits should be imposed
except as set forth in the following claims.
* * * * *